Sanfilippo Syndrome B is an autosomal recessive lysosomal storage disease (LSD) caused by a deficiency in a lysosomal enzyme known as N-acetyl-alpha-D-glucosaminidase (NaGlu). NaGlu is required for the degradation of heparan sulfate as part of the stepwise breakdown of glycosaminoglycans (GAG) in the lysosome. The deficiency or absence of NaGlu leads to accumulation and urinary excretion of heparan sulfate. With over 70 different mutations identified to date, Sanfilippo Syndrome B exhibits extensive molecular and genetic heterogeneity.
Approximately 1 out of 200,000 births is affected by Sanfilippo Syndrome B and the deficiency mainly manifests in young children. After initial symptom-free interval, patients suffering from Sanfilippo Syndrome B usually present with a slowing of mental development and behavioral problems, followed by progressive intellectual decline resulting in severe mental retardation, dementia and motor disease. Acquisition of speech is slow and incomplete. Profoundly affected patients may present delayed psychomotor and speech development as early as 2 years of age. The disease usually progresses to increasing behavioral disturbance and sleep disturbance. Although the clinical features are mainly neurological, patients often develop diarrhea, carious teeth, an enlarged liver and spleen, stiff joints, hirsteness and/or coarse hair and may exhibit blood-clotting problems. In the final stage of the illness, patients become immobile and unresponsive and develop swallowing difficulties and seizure. The lifespan of an affected child typically does not extend beyond late teens to early twenties.
Different approaches have been attempted to provide the missing enzyme in patients. To produce NaGlu for enzyme replacement therapy (ERT), human NaGlu has been expressed in various mammalian cell culture systems. However, in contrast to the naturally occurring NaGlu which trafficks to the lysosome intracellularly, recombinant NaGlu proteins produced and secreted from mammalian cells were found to contain no or only a trace amount of mannose 6-phosphate (M6P). The absence or scarcity of M6P moieties in the secreted NaGlu has been known to prevent its efficient internalization into target cells (e.g., human skin fibroblasts), which have M6P receptors on the surface on its plasma membrane (see, Zhao et al., Protein Expression and Purification, 19:202-211 (2000); and Weber et al., Protein Expression and Purification, 21:251-259 (2001)). The low degree of phosphorylation was seen in secreted mouse NaGlu expressed in CHO cells, secreted human NaGlu expressed in HeLa cells, secreted human NaGlu expressed in human fibroblasts, and secreted human NaGlu expressed in human embryonic kidney (HEK) cell line 293 (see, Zhao et al., Protein Expression and Purification, 19:202-211 (2000); Yogalingam et al., Biochim Biophys. Acta 1502: 415-425; and Weber et al., Protein Expression and Purification, 21:251-259 (2001)). No or weak phosphorylation of N-glycans in the NaGlu proteins secreted from the mammalian cells has posed a major obstacle for the development of a recombinant human NaGlu protein suitable for enzyme replacement therapy as all the aforementioned attempts has failed to produce an enzyme which is efficiently taken up by target cells as the concentration of the internalized proteins, if detectable at all, was nearly a thousand times less than wild-type levels (see, Zhao et al., Protein Expression and Purification, 19:202-211 (2000)). To date, no approved product is available for the treatment of Sanfilippo Syndrome B.
Direct administration of mammalian cell-produced recombinant human NaGlu protein (rhNaGlu) having the native amino acid sequence into the central nervous system (CNS) (e.g., intrathecal administration into the cerebrospinal fluid (CSF)) of NaGlu deficient mice has been attempted, but failed to demonstrate successful biodistribution of the enzyme to the brain due to excessive accumulation of the protein on the ependymal ling of the ventricles as well as lack of requisite M6P residues for efficient cellular uptake. Similarly, systemic administration (i.e., intravenous (IV) injection) of mammalian cell-produced rhNaGlu having the native amino acid sequence also failed to demonstrate successful localization of the protein to the brain. In addition to known risks associated with highly invasive intrathecal administration, these obstacles in targeting rhNaGlu to the brain have been too great a challenge to achieve effective therapy for the treatment of Sanfilippo Syndrome B.
Therefore, there is a need to provide a stable NaGlu protein which is enzymatically active and has physical properties that allow for the protein to cross the blood brain barrier (BBB) and for effective internalization of the protein into the lysosomes of target cells. There is also a need for a high expressing and robust protein production platform which can provide a recombinant human NaGlu that effectively crosses the blood brain barrier and is efficiently internalized into human target cells.